Direct Observation of Torsional Motion of Group II Chaperonin

Abstract

Diffracted X-ray Tracking (DXT) has been considered as a powerful technique in biological science for detecting subtle (pico meter scale) dynamic motion of the target protein at single molecular level. This method was applied for various proteins, such as bacteriorhodopsin [1], antibody [2] and KcsA channel [3]. In DXT, the dynamics of a single protein can be monitored through trajectory of the Laue spot from the nanocrystal which was labeled on the objective protein immobilized on the substrate surface.In this study, DXT method was applied to the group II chaperonin, a protein machinery that captures an unfolded protein and refolds it to the correct conformation in an ATP dependent manner [4]. A mutant group II chaperonin from Thermococcus strain KS-1 with a Cys residue at the tip of the helical protrusion, was immobilized on the gold coated substrate surface and was labeled with a gold nanocrystal through gold-thiol bond.We monitored diffracted spots from the nanocrystal as dynamic motion of the chaperonin, and found that the rotational motion of the nanocrystal, which corresponded to the torsional motion of the chaperonin, in the presence of ATP condition was 10 times larger than that in the absence of ATP condition. And UV-light triggered DXT study using caged ATP revealed that the chaperonin twisted counterclockwisely (from the top to the bottom view of chaperonin) when ATP binded to the chaperonin and the angular velocity from open to closed state of chaperonin chamber was 10 % faster than that from closed to open state.[1] Y. Okumura et al., Phys. Rev. E, 70:021917 (2004)[2] T. Sagawa et al., Biochem. Biophys. Res. Commun. 335:770 (2007)[3] H. Shimizu et al., Cell 132:67 (2008)[4] T. Kanzaki et al., J. Biol. Chem. 283: 34773 (2008)